Elaina M. Tuttle

The value of avian genomics to the conservation of wildlife.

BackgroundGenomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution.Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care).In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes.ResultsGenotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current populations genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology.Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one.ConclusionMining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease.In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

PROMISCUITY DRIVES SEXUAL SELECTION IN A SOCIALLY MONOGAMOUS BIRD

Abstract Many socially monogamous species paradoxically show signs of strong sexual selection, suggesting cryptic sources of sexual competition among males. Darwin argued that sexual selection could operate in monogamous systems if breeding sex ratios are biased or if some males attract highly fecund females. Alternatively, sexual selection might result from promiscuous copulations outside the pair bond, although several recent studies have cast doubt on this possibility, in particular by showing that variance in apparent male reproductive success (number of social young) differs little from variance in actual male reproductive success (number of young sired). Our results from a long-term study of the socially monogamous splendid fairy-wren (Malurus splendens) demonstrate that such comparisons are misleading and do not adequately assess the effects of extra-pair paternity (EPP). By partitioning the opportunity for selection and calculating Bateman gradients, we show that EPP has a strong effect on male annual and lifetime fitness, whereas other proposed mechanisms of sexual selection do not. Thus, EPP drives sexual selection in this, and possibly other, socially monogamous species.

Genetic similarity of social mates predicts the level of extrapair paternity in splendid fairy-wrens

Why females mate with multiple males, particularly in socially monogamous species, is one of the central unanswered questions in sexual selection and behavioural ecology. Recent theory suggests that socially monogamous females may improve the genetic quality of their offspring by mating with extrapair males with whom they are more genetically dissimilar (relative to their social mates), because decreased genetic similarity between mates may lead to decreased inbreeding depression of offspring and other benefits. We found that levels of extrapair paternity in splendid fairy-wren, Malurus splendens, broods were predicted by genetic similarity between social mates. Females whose entire broods were sired by extrapair males were more genetically similar to their social mates than they were to their extrapair mates, but females were no less similar to their extrapair mates than they were to the average male from the population. Within-pair young (WPY) from mixed-paternity broods showed higher levels of inbreeding and lower heterozygosity than did their extrapair nestmates or WPY from broods without extrapair paternity. Several processes could account for these patterns, including female choice of genetically dissimilar mates, differential sperm investment by males or differential survival of embryos as a function of genetic dissimilarity between mates, or postcopulatory processes such as sperm competition, cryptic female choice, or interactions between sperm and ova. Regardless of the mechanism, our results suggest that female splendid fairy-wrens may indirectly benefit from extrapair mating because it leads to lower levels of inbreeding and increased heterozygosity in their offspring.

Cloacal protuberances and extreme sperm production in Australian fairy-wrens

In many passerine species males have enlarged cloacal protuberances during the breeding season. One hypothesis for the evolution of cloacal protuberances posits that they are a response to sperm competition and thus predicts that both within and between species the size of the protuberance correlates with the number of sperm stored. Here we provide the first intraspecific test of this hypothesis. In Australian fairy-wrens (Aves: Maluridae) females regularly mate outside of their social group resulting in intense sperm competition among males. Male fairy-wrens develop enlarged cloacal protuberances, and in a study of three species, splendid fairy-wren, white-winged fairy-wren, and variegated fairy-wren, we found significant intraspecific correlations between the size of a male’s protuberance and the stored sperm reserves in two of the three species. Males of these species had extreme numbers of sperm in their cloacal protuberances, up to 8.3 billion for splendid fairy-wrens, which should be available for a single ejaculate and is the most ever reported in an avian species. Studies of both captive and wild males showed that individuals can produce as many as 2 billion sperm per day. These data support the sperm competition hypothesis for the evolution of cloacal protuberances and highlight extreme sperm production as one possible outcome of sperm competition.

SPATIAL DYNAMICS OF ALTERNATIVE REPRODUCTIVE STRATEGIES: THE ROLE OF NEIGHBORS

In territorial species, the reproductive success of a male is dependent on the quality of his territory. One important component of territory quality is spatial location. High-quality territories not only should be located in areas of high food abundance and low predation, but also should be located in areas that offer optimal amounts of social interaction. Such optima might be different for individuals according to their sex, domi- nance, or genotype. We studied territory quality (size, vegetation structure, and placement) in a socially monogamous, polymorphic passerine, the White-throated Sparrow (Zonotrichia albicollis), in order to determine how spatial attributes contribute to selection intensity on two genotypes. In this species, plumage (white and tan), behavior, and life-history char- acteristics have a genetic basis and are correlated with the presence or absence of a chro- mosomal inversion. Using remote sensing and Geographic Information Systems (GIS), we found that the territories of white and tan males do not differ in size or vegetation structure, suggesting that these factors are not of primary importance to males when deciding where to establish a territory. Instead, we suggest that the placement of white and tan territories depends on the number of neighbors (particularly, white male neighbors). Tan males settle in low-density, neighbor-restrictive habitats where intruder pressure from white males seek- ing extra-pair copulations is reduced. In contrast, white males tend to settle in high-density areas where the probability of encountering neighboring fertile females is greatest. This segregation has led to intraspecific niche partitioning in the two disassortative pair types so that each male morph can best exploit its respective reproductive strategies. These factors may, in turn, contribute to the maintenance of this unusual mating system and, ultimately, the stability of the polymorphism in this species. Similar forces may be operating in other species without distinct morphological markers; we suggest that researchers keep social factors in mind when examining habitat selection.

Examining the social landscapes of alternative reproductive strategies

Social environments are inherently dynamic, often changing depending on the frequency and outcomes of conspecific interactions – they can be simultaneously the targets and agents of selection. Understanding how organisms settle in heterogeneous social environments and the effects this has on reproductive success is vital to our understanding of the selective forces at work in wild populations. From an intensive behavioural and ecological study of territoriality in the polymorphic white‐throated sparrow (Zonotrichia albicollis), we demonstrate that males of the two morphs inhabit social niches that correspond to their respective alternative reproductive strategies. By integrating molecular ecology and Geographic Information Systems, we generated continuous, socio‐spatial models of local conspecific density and cuckoldry risk. Our results suggest that the morphs segregate their territories based on socio‐spatial variables, creating a heterogeneous social landscape that matches each behavioural phenotype with a favourable social environment. Specifically, the monogamous tan males tended to settle in low‐density areas that were also low for cuckoldry risk, while the opposite was true for the promiscuous white males. This pattern of socio‐spatial heterogeneity, combined with the social niche partitioning we observed, might act as a social niche polymorphism, and play an important role in maintenance of the alternative reproductive strategies of the white‐throated sparrow. Socio‐spatial factors, similar to those observed in the white‐throated sparrow, may play important roles in the evolution of mating systems in other species, even those with more continuous or cryptic variation.

Brain transcriptome sequencing and assembly of three songbird model systems for the study of social behavior

Emberizid sparrows (emberizidae) have played a prominent role in the study of avian vocal communication and social behavior. We present here brain transcriptomes for three emberizid model systems, song sparrow Melospiza melodia, white-throated sparrow Zonotrichia albicollis, and Gambel’s white-crowned sparrow Zonotrichia leucophrys gambelii. Each of the assemblies covered fully or in part, over 89% of the previously annotated protein coding genes in the zebra finch Taeniopygia guttata, with 16,846, 15,805, and 16,646 unique BLAST hits in song, white-throated and white-crowned sparrows, respectively. As in previous studies, we find tissue of origin (auditory forebrain versus hypothalamus and whole brain) as an important determinant of overall expression profile. We also demonstrate the successful isolation of RNA and RNA-sequencing from post-mortem samples from building strikes and suggest that such an approach could be useful when traditional sampling opportunities are limited. These transcriptomes will be an important resource for the study of social behavior in birds and for data driven annotation of forthcoming whole genome sequences for these and other bird species.

Genes located in a chromosomal inversion are correlated with territorial song in white-throated sparrows

The genome of the white‐throated sparrow (Zonotrichia albicollis) contains an inversion polymorphism on chromosome 2 that is linked to predictable variation in a suite of phenotypic traits including plumage color, aggression and parental behavior. Differences in gene expression between the two color morphs, which represent the two common inversion genotypes (ZAL2/ZAL2 and ZAL2/ZAL2m), may therefore advance our understanding of the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified gene expression and terrirorial aggression, including song, in a population of free‐living white‐throated sparrows. We analyzed gene expression in two brain regions, the medial amygdala (MeA) and hypothalamus. Both regions are part of a ‘social behavior network’, which is rich in steroid hormone receptors and previously linked with territorial behavior. Using weighted gene co‐expression network analyses, we identified modules of genes that were correlated with both morph and singing behavior. The majority of these genes were located within the inversion, showing the profound effect of the inversion on the expression of genes captured by the rearrangement. These modules were enriched with genes related to retinoic acid signaling and basic cellular functioning. In the MeA, the most prominent pathways were those related to steroid hormone receptor activity. Within these pathways, the only gene encoding such a receptor was ESR1 (estrogen receptor 1), a gene previously shown to predict song rate in this species. The set of candidate genes we identified may mediate the effects of a chromosomal inversion on territorial behavior.

Variation in Preen Oil Composition Pertaining to Season, Sex, and Genotype in the Polymorphic White-Throated Sparrow

Evidence for the the ability of birds to detect olfactory signals is now well documented, yet it remains unclear whether birds secrete chemicals that can be used as social cues. A potential source of chemical cues in birds is the secretion from the uropygial gland, or preen gland, which is thought to waterproof, maintain, and protect feathers from ectoparasites. However, it is possible that preen oil also may be used for individual recognition, mate choice, and signalling social/sexual status. If preen oil secretions can be used as socio-olfactory signals, we should be able to identify the volatile components that could make the secretions more detectable, determine the seasonality of these secretions, and determine whether olfactory signals differ among relevant social groups. We examined the seasonal differences in volatile compounds of the preen oil of captive white-throated sparrows, Zonotrichia albicollis. This species is polymorphic and has genetically determined morphs that occur in both sexes. Mating is almost exclusively disassortative with respect to morph, suggesting strong mate choice. By sampling the preen oil from captive birds in breeding and non-breeding conditions, we identified candidate chemical signals that varied according to season, sex, morph, and species. Linear alcohols with a 10–18 carbon chains, as well as methyl ketones and carboxylic acids, were the most abundant volatile compounds. Both the variety and abundances of some of these compounds were different between the sexes and morphs, with one morph secreting more volatile compounds in the non-breeding season than the other. In addition, 12 compounds were seasonally elevated in amount, and were secreted in high amounts in males. Finally, we found that preen oil signatures tended to be species-specific, with white-throated sparrows differing from the closely related Junco in the abundances and/or prevalence of at least three compounds. Our data suggest roles for preen oil secretions and avian olfaction in both non-social as well as social interactions.

ELOPTA: a novel microcontroller-based operant device.

Operant devices have been used for many years in animal behavior research, yet such devices are generally highly specialized and quite expensive. Although commercial models are somewhat adaptable and resilient, they are also extremely expensive and are controlled by difficult to learn proprietary software. As an alternative to commercial devices, we have designed and produced a fully functional, programmable operant device, using a PICmicro microcontroller (Microchip Technology, Inc.). The electronic operant testing apparatus (ELOPTA) is designed to deliver food when a study animal, in this case a bird, successfully depresses the correct sequence of illuminated keys. The device logs each keypress and can detect and log whenever a test animal is positioned at the device. Data can be easily transferred to a computer and imported into any statistical analysis software. At about 3% the cost of a commercial device, ELOPTA will advance behavioral sciences, including behavioral ecology, animal learning and cognition, and ethology.